15N Stable Isotope Labeling PSTs in Alexandrium minutum for Application of PSTs as Biomarker - MDPI
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toxins Article 15 N Stable Isotope Labeling PSTs in Alexandrium minutum for Application of PSTs as Biomarker Wancui Xie 1,2 , Min Li 1 , Lin Song 1,2 , Rui Zhang 3 , Xiaoqun Hu 1 , Chengzhu Liang 4 and Xihong Yang 1,2, * 1 College of Marine Science and Biological Engineering, Qingdao University of Science & Technology, Qingdao 266042, China; xiewancui@163.com (W.X.); 2017170007@mails.qust.edu.cn (M.L.); lylinsong@hotmail.com (L.S.); 2018170002@mails.qust.edu.cn (X.H.) 2 Key Laboratory for Biochemical Engineering of Shandong Province, Qingdao 266042, China 3 College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, China; zhangr1168@163.com 4 Shandong Entry-Exit Inspection and Quarantine Bureau, Qingdao 266000, China; liangcz@163.com * Correspondence: 03137@qust.edu.cn Received: 27 February 2019; Accepted: 4 April 2019; Published: 8 April 2019 Abstract: The dinoflagellate Alexandrium minutum (A. minutum) which can produce paralytic shellfish toxins (PSTs) is often used as a model to study the migration, biotransformation, accumulation, and removal of PSTs. However, the mechanism is still unclear. To provide a new tool for related studies, we tried to label PSTs metabolically with 15 N stable isotope to obtain 15 N-PSTs instead of original 14 N, which could be treated as biomarker on PSTs metabolism. We then cultured the A. minutum AGY-H46 which produces toxins GTX1-4 in f/2 medium of different 15 N/P concentrations. The 15 N-PSTs’ toxicity and toxin profile were detected. Meanwhile, the 15 N labeling abundance and 15 N atom number of 15 N-PSTs were identified. The 14 N of PSTs produced by A. minutum can be successfully replaced by 15 N, and the f/2 medium of standard 15 N/P concentration was the best choice in terms of the species’ growth, PST profile, 15 N labeling result and experiment cost. After many (>15) generations, the 15 N abundance in PSTs extract reached 82.36%, and the 15 N atom number introduced into GTX1-4 might be 4–6. This paper innovatively provided the initial evidence that 15 N isotope application of labeling PSTs in A. minutum is feasible. The 15 N-PSTs as biomarker can be applied and provide further information on PSTs metabolism. Keywords: Alexandrium minutum; dinoflagellate; paralytic shellfish toxins (PSTs); 15 N stable isotope labeling; biomarker Key Contribution: The 14 N of paralytic shellfish toxins produced by experimental Alexandrium minutum can be replaced by 15 N successfully. 15 N abundance in paralytic shellfish toxins extract reached 82.36%, and 15 N atom number introduced into Gonyautoxin1-4 may be 4–6. 15 N-paralytic shellfish toxins as biomarker have potential application in some fields such as metabolomics and clinical mechanism of paralytic shellfish toxins. 1. Introduction Harmful algal blooms (HABs) occur frequently in coastal areas worldwide, causing public concerns. Firstly, HABs cause millions of dollars economic losses in the tourism and industry sectors [1]. Secondly, HABs break the balance of marine ecosystems as they can disrupt communities and food web structures [2,3]. Thirdly, phycotoxins produced by HABs may be transferred through the food cycle, thereby causing lethal and sublethal effects on humans [4,5]. Among all the toxins produced by HABs Toxins 2019, 11, 211; doi:10.3390/toxins11040211 www.mdpi.com/journal/toxins
Toxins 2019, 11, 211 2 of 13 species, paralytic shellfish toxins (PSTs) produced by dinoflagellates are the most widespread and potent shellfish contaminating biotoxins [6]. They are accumulated by filter-feeding bivalve mollusks and some zoophagous mollusks [7]. One of the most widespread toxigenic microalgal taxa is the dinoflagellate Alexandrium, and studies have demonstrated that toxic Alexandrium spp. disrupt behavioral and physiological processes in marine filter feeders [8,9]. In recent years, A. tamarense, A. minutum, A. ostenfeldii, and others were used as carriers of PSTs to track the migration, biotransformation, accumulation, and removal of PSTs under experimental conditions [10–12]. However, the metabolism of PSTs in bivalves is still unclear. Thus, there is a need for a new powerful tool to conduct PST-related researches. Isotopic tracer technique can be used to study the absorption, distribution, metabolism, and excretion of some substances, such as protein and lipid, or to establish organism trophic links in the ecosystem [13–16]. The stable isotope labeling (ICAT, ICPL, IDBEST, iTRAQ, TMT, IPTL, and SILAC)-based quantitative approaches are highly efficient for obtaining highly accurate quantification results and for building more extensive small molecule databases [14,17], especially in proteomics [18,19]. Some studies were conducted on microalgae due to their ability to fix stable isotopes photosynthetically into cells and offer various target products [20–23]. Artificially enriched stable isotopes of nitrogen can be fed to cells, either in the form of 15 N-labeled amino acid or 15 N-labeled inorganic salt. According to [24], the microalgae Chlamydomonas reinhardtii was used to produce 15 N-labeled amino acids with a high isotopic enrichment and sixteen 15 N-labelled amino acids could be obtained. However, isotope-labeling toxin-producing algae have been rarely studied so far. In the present study, we metabolically labeled the A. minutum produced gonyautoxins1-4 (GTX1-4) that belonging to mono-sulfated subgroup of PSTs [25]. The culture was carried out with 15 N-NaNO3 as nitrogen source to obtain the 15 N-PSTs that could be used as biomarker and provide further information on PSTs metabolism in filter feeding bivalve. 2. Results 2.1. Batch Culture: 15 N/P Influence in Algal Growth and Toxicity Initial batch culture experiments allowed comparison of algae growth under different 15 N/P conditions, as follows: Group A: 1.0 time of the f/2 medium standard 15 N/P concentration; Group B: 1.5 times of the f/2 medium standard 15 N/P concentration; Group C: 2.0 times of the f/2 medium standard 15 N/P concentration; Group D: 2.5 times of the f/2 medium standard 15 N/P concentration; and Group E: 3.0 times of the f/2 medium standard 15 N/P concentration. The results showed that 15 N/P concentration can affect algal growth (Figure 1a). In an optimal culture environment, the growth curve of algae cell can be roughly divided into 4 phases, as follows: lag phase 0–15 days; log phase 16–30 days; 31 days of the stable phase; and decay phase. In this study, the decay phase was not studied, because the focus was on the degree of 15 N labeling of algal cellular toxin. The results were similar to those obtained in the report [26]. However, the lag phase of this experiment was much longer, and it may have been caused by partial mechanical damage to cells when algal cells were collected by centrifugation. After the growth of algal cells into the log phase, Group B cell density was higher than those of the other four groups (p < 0.05). At 30 days, the maximum value of Group B reached 3.820 × 104 cells/mL, and there was no significant difference in Group A cell density (3.342 × 104 cells/mL) (p > 0.05). However, the biomass of Groups A and B biomass was much higher than that of the other three groups (p < 0.01). Lower biomass at high nutrient concentration could be attributed to the inhabitation of photosystem II’s photosynthetic capacity at high nutrient levels [27]. 15 N/P conditions also can affect toxicity of algae cells (Figure 1b). The highest toxin levels were determined at day 25 in the log phase in all groups, the same as [28], not early- or post- stationary growth phase mentioned in [29,30]. Concerning the factors influencing toxicity, the toxin content per cell in batch culture was not only related to the cell growth stage, but was also affected by intracellular
Toxins 2019, 11, 211 3 of 13 Toxins 2019, 11, 211 3 of 12 nutrient salts (e.g., nitrogen, phosphorus, and carbon dioxide), thereby reflecting the balance between Toxins 2019, 11,and synthesis 211 leakage of toxins (e.g., catabolism and cell division) [31]. 3 of 12 (a) (b) (a) (b) Figure 1. The effects of 15N/P concentration on cell growth (a) and toxicity (b) in batch culture. (——, 15 N/P −−−−, Figure Figure TheThe ∙∙∙∙∙∙∙∙, 1. 1. −∙−∙−∙effects effects and of of 15−∙∙−∙∙−∙∙: concentration on cellongrowth Growth/Sigmoidal. N/P concentration cell growth Letters and(a) (a)indicate and toxicity significant toxicity (b) in batchbetween differences (b) in batch culture. culture. (——, (——, −−−−, conditions). ········, −·−·−· and −··−··−··: Growth/Sigmoidal. Letters indicate significant −−−−, ∙∙∙∙∙∙∙∙, −∙−∙−∙ and −∙∙−∙∙−∙∙: Growth/Sigmoidal. Letters indicate significant differences between differences between conditions). conditions). 2.2. Generation to Generation Culture: 1515 N/P Influence in Algal Growth and Toxicity 2.2. Generation to Generation Culture:15 N/P Influence in Algal Growth and Toxicity 2.2. Generation The algaltocells Generation Culture: of Groups A and N/PBInfluence in Algal were chosen viaGrowth and Toxicity generation to generation culture (three The algal cells of Groups A and B were chosen via generation to generation culture (three generations) The algalbecause cells ofofGroups better growth. A and BThe effects were of 15N/P chosen 15 via concentration generation to on cell growth generation and toxicity culture (three generations) because of better growth. The effects of15 N/P concentration on cell growth and toxicity are displayed in Figure 2. In the first generation, Group B algae cell growth generations) because of better growth. The effects of N/P concentration on cell growth and toxicity was better than that of are displayed in Figure 2. In the first generation, Group B algae cell growth was better than that Group A; the same results were obtained in batch culture experiments. are displayed in Figure 2. In the first generation, Group B algae cell growth was better than that In the second and third of of Group A; the same results were obtained in batch culture experiments. In the second and third generations, Group A; the the same cellresults density of Group were obtained B was less culture in batch than that of Group A, experiments. In especially the secondinand thethird third generations, the cell density of Group B was less than that of Group A, especially in the third generation. generation. Interestingly, generations, the cell density there of was Group no significant B was lessdifference than that (pof< Group 0.05) inA, algal cytotoxicity especially in the between third Interestingly, there was no significant difference (p < 0.05) in algal cytotoxicity between the two groups the two groups generation. in all three Interestingly, generations. there The experimental was no significant difference results showed (p < 0.05) the algae in algal cells cultured cytotoxicity betweenin in all three generations. The experimental results showed the algae cells cultured in a nutrient solution a nutrient the solution two groups in all with threeagenerations. higher 15N/P than The f/2 medium experimental standard results showed15N/P theconcentration gradually algae cells cultured in with a higher 15 N/P than f/2 medium standard 15 N/P concentration gradually deteriorated, indicating deteriorated, indicating a nutrient solution with a higher that f/2 medium 15 standard 15N/P concentration was N/P than f/2 medium standard N/P concentration gradually 15 more suitable for that f/2 medium standard 15 N/P concentration was more suitable for domesticating high abundance domesticating deteriorated, high abundance indicating 15 N-PST A. minutum. that f/2 N-PST 15 medium A. minutum. standard 15N/P concentration was more suitable for domesticating high abundance N-PST A. minutum. 15 (a) (b) Figure 2.2. Effects (a) Effectsofof1515 N/Pconcentration concentrationononcell cellgrowth growth(a) (a)and andcell celltoxicity toxicity(b) (b)in (b) in generation generation to to Figure N/P generationculture. generation culture. Figure 2. Effects of 15N/P concentration on cell growth (a) and cell toxicity (b) in generation to 15 N/P Effect 2.3.generation culture. on Algae PSTs Profile 2.3. 15N/P Effect on Algae PSTs Profile The experiment 2.3. 15N/P was carried out using 1.0 and 1.5 times of the f/2 medium standard 15 N/P The Effect on Algaewas experiment PSTs Profile out carried using 1.0 and 1.5 times of the f/2 medium 15 N/Pstandard 15N/P concentration compared with 1.0 and 1.5 times of the f/2 medium standard concentration. concentration compared with 1.0 and 1.5 times 1.0of the 1.5 f/2 medium of standard 15N/P concentration. The TheThe experiment A.was carried outproduced using and times the f/2 mediumare standard 15N/P experimental minutum only GTX1-4; the major components GTX-2,-3, which experimental concentration A. minutum compared withonly produced GTX1-4; the major components are GTX-2,-3, which of1.0 theand 1.5toxin times(Figure of the f/2 3).medium standard N/P concentration. The 15 accounted for about 74% total Changing the standard N/P concentration accounted forA.about experimental 74% of minutum theproduced only total toxinGTX1-4; (Figure the 3). Changing major the standard components are N/P concentration GTX-2,-3, which influenced the profile of PSTs, and no significant difference was found between 1.0 time of the f/2 influencedfor accounted theabout profile of PSTs, 74% of theand totalnotoxin significant (Figuredifference was found 3). Changing betweenN/P the standard 1.0 concentration time of the f/2 medium standard 15N/P and 14N/P concentration, indicating that the application of 15N isotope influenced the profile of PSTs, and no significant difference was found between 1.0 time of the f/2 labeling can medium feasibly standard be used 15N/P and to studyconcentration, 14N/P PSTs production and metabolism. indicating that the application of 15N isotope labeling can feasibly be used to study PSTs production and metabolism.
Toxins 2019, 11, 211 4 of 13 medium standard 15 N/P and 14 N/P concentration, indicating that the application of 15 N isotope labeling Toxins 2019, 11, 211 4 of 12 can feasibly be used to study PSTs production and metabolism. Figure 3. The effects Figure 3. of 15 effects of 15N/P concentration on A. minutum PST profile. 2.4. 15 N Labeling Abundance Change of 15 N-PSTs 2.4. 15N Labeling Abundance Change of 15N-PSTs The 15 N labeling abundance was calculated according to the following formula: The 15N labeling abundance was calculated according to the following formula: 15 N atom% = 1/(2I28 /I29 + 1) × 100 (15 N atom% < 10%); 15N atom% 15 N atom% = 2/(I /I= 1/(2I28/I29+1) ×15100 (15N atom% 10%) (1) 15N atom% = 2/(I29/I30+2) × 100 (15N atom%>10%) In the algal culture environment, 15 N-NaNO3 of f/2 medium is not the only nitrogen source because of the inorganic 14 N existing in15natural seawater. A. minutum cells prefer absorbing light In the algal culture environment, N-NaNO3 of f/2 medium is not the only nitrogen source elements 14 ( the N) and rejecting14Nheavy elements (15 N), resultingA.in minutum nitrogen stable isotope fractionation. because of inorganic existing in natural seawater. cells prefer absorbing light With increasing cell density and size, less and less 14 N can be utilized. Thus, more 15 N can enter elements ( N) and rejecting heavy elements ( N), resulting in nitrogen stable isotope fractionation. 14 15 cells, With and nitrogen increasing stable cell isotopes density fractionation and size, less and lessweakens. 14N canTwo culture methods be utilized. Thus, more and15N gascan isotope enter mass cells, spectrometer were used to determine the relationship between 15 N-labeling abundance and culture and nitrogen stable isotopes fractionation weakens. Two culture methods and gas isotope mass time, as shownwere spectrometer in Table used1.toDuring the batch determine culture, 15 Nbetween the relationship abundance was positively 15N-labeling related abundance to culture and culture time time,and reached as shown in the highest Table valuethe 1. During at batch day 30. There 15are culture, significant differences N abundance (p 90%). below the abundance of the labeling material 15N- NaNO3 (δ15N = 99.14%), not reaching our expectation (>90%). Table 1. 15 N labeling abundance change of PSTs along with the change of culture time in different cultivation Table 1. 15Nmethods. labeling abundance change of PSTs along with the change of culture time in different cultivation methods. 1.0 Time of the f/2 1.5 Times of the f/2 Medium Standard Medium Standard Culture Method 1.0 Time 15 N/Pof the f/2 1.5 Times 15 N/Pof the f/2 Concentration Concentration Medium Standard Medium Standard Culture Method 15N/P Concentration 15 PSTs N Abundance (Atom%) 15N/P Concentration 0 PSTs 150.47 N Abundance (Atom%) 0.47 Batch culture/d 20 26.26 26.43 0 0.47 0.47 30 57.16 70.26 Batch culture/d 1 20 26.26 37.60 26.43 - Generation to generation 2 30 57.16 58.32 70.26 - culture/generation 3 1 37.60 62.46 - - Generation to generation - No. n generation 82.36 2 58.32 - culture/generation 3 62.46 - No. n generation - 82.36 2.5. The Efficiency of 15N-PSTs Separation and Purification N-PSTs extracts were separated and purified by column chromatography on the Bio-Gel P-2 15 and the weak cation exchanger Bio-Rex 70. In the process of column chromatography on the Bio-Gel P-2, fluorescence detection and UV absorbance detection were carried out (Figure 4a). The UV
Toxins 2019, 11, 211 5 of 13 2.5. The Efficiency of 15 N-PSTs Separation and Purification 15 N-PSTs extracts were separated and purified by column chromatography on the Bio-Gel P-2 and Toxins 2019, 11, 211 5 of 12 the weak cation exchanger Bio-Rex 70. In the process of column chromatography on the Bio-Gel P-2, fluorescence detection and UV absorbance detection were carried out (Figure 4a). The UV absorption absorption peak did not coincide with the fluorescence absorption peak. Thus, the UV absorption peak did not coincide with the fluorescence absorption peak. Thus, the UV absorption signal had signal had no relationship with the toxin component. As shown by the fluorescence absorption peak, no relationship with the toxin component. As shown by the fluorescence absorption peak, Bio-Gel Bio-Gel P-2 effectively separated 15 15N-PSTs with impurities in crude extracts, such as proteins and P-2 effectively separated N-PSTs with impurities in crude extracts, such as proteins and pigments. pigments. The liquid of fluorescence absorption peak was collected, freeze-dried (10 mg), and The liquid of fluorescence absorption peak was collected, freeze-dried (10 mg), and redissolved redissolved with 0.05 M Tri-HCl (2 mL). The redissolved sample (1 mL) was used for purification by with 0.05 M Tri-HCl (2 mL). The redissolved sample (1 mL) was used for purification by column column chromatography on weak cation exchanger Bio-Rex 70 at gradient elution condition and was chromatography on weak cation exchanger Bio-Rex 70 at gradient elution condition and was separated separated (Figure (Figure 4b). After 4b). Afterthe analysis, analysis, Peak I the wasPeak Ⅰ was determined determined to be the to be the isomer isomer mixture of mixture GTX1/4, of andGTX1/4, Peak II and Peak Ⅱ was the isomer mixture was the isomer mixture of GTX2/3. of GTX2/3. (a) (b) Gonyautoxin1-4purified Figure4.4.Gonyautoxin1-4 Figure purifiedby bycolumn columnchromatography chromatographyon onthe theBio-Gel Bio-GelP-2 P-2(a) (a)and andthe theweak weak cation exchanger BioRex 70 (b). cation exchanger BioRex 70 (b). 2.6. 15 N Atom Number Identification of 15 N-PSTs 2.6. 15N Atom Number Identification of 15N-PSTs As demonstrated previously [32], GTX1-4 (Figure 5) can be ionized in ESI positive ion mode, As demonstrated previously [32], GTX1-4 (Figure 5) can be ionized in ESI positive ion mode, thereby giving abundant fragment ions (Table 2). When 14 N of PSTs is replaced by 15 N, [M+H]+ thereby giving abundant fragment ions (Table 2). When 14N of PSTs is replaced by 15N, [M+H]+ and and fragment ions will change with the number of 15 N atoms, so that primary mass spectrum can fragment ions will change with the number of N atoms, so that primary mass spectrum can be used 15 be used to determine the 15 N atom number of 15 N-PSTs. After the addition of artificial nitrogen in to determine the 15N atom 15 number of 15N-PSTs. After the addition of artificial nitrogen in A. minutum A. minutum culture, N was successfully introduced to PSTs, and characteristic fragment ions were culture, 15N was successfully introduced to PSTs, and characteristic fragment ions were produced. produced. There was no [M+H]+ peak in the mass spectrum as the result of high capillary voltage There was no [M+H] peak in the mass spectrum as the result of high capillary voltage in this + in this experiment (Figure 6). GTX1 produced three characteristic fragment ions (m/z = 334.2966; experiment (Figure 6). GTX1 produced three characteristic fragment ions (m/z=334.2966; 318.3007; 318.3007; 319.3054) with the loss of -SO3 or -SO3 -H2 O. GTX4 generated three characteristic fragment 319.3054) with the loss of -SO3 or -SO3-H2O. GTX4 generated three characteristic fragment ions ions (m/z = 318.3007; 319.3054; 338.3418) with the loss of -SO3 -H2 O or -SO3 and had two fragment (m/z=318.3007; 319.3054; 338.3418) with the loss of -SO3-H2O or -SO3 and had two fragment ions ions (m/z = 318.3007; 319.3054) similar to those of GTX1. There were four characteristic fragment ions (m/z=318.3007; 319.3054) similar to those of GTX1. There were four characteristic fragment ions (m/z = 322.1197; 321.1252; 305.1583; 317.0497) in GTX2 with the loss of -SO3 or -SO3 -H2 O, whereas (m/z=322.1197; 321.1252; 305.1583; 317.0497) in GTX2 with the loss of -SO3 or -SO3-H2O, whereas two two characteristic fragment ions (m/z = 302.3054; 303.3095) were obtained with the loss of -SO3 -H2 O. characteristic fragment ions (m/z=302.3054; 303.3095) were obtained with the loss of -SO3-H2O. Theoretically, the m/z of fragment ions can increase (+1,+2,+3,+4,+5,+6,+7) corresponding with the Theoretically, the m/z of fragment ions can increase (+1,+2,+3,+4,+5,+6,+7) corresponding with the number (1,2,3,4,5,6,7) of introduced 15 N atoms in PSTs. Based on these data, 2~6 15 N atoms can be number (1,2,3,4,5,6,7)15of introduced N atoms in15PSTs. Based on these data, 2~6 15N atoms can be 15 introduced into the N-PSTs molecule, and 4~6 N atoms is most possible. introduced into the 15N-PSTs molecule, and 4~6 15N atoms is most possible. Table 2. Mass spectral data for gonyautoxins (GTX1-4). Molecular Fragment Ion after Toxin [M+H]+ Fragment Ion Loss of Formula Labeling GTX1 C10H17N7O9S 412 332; 314 -SO3; -SO3-H2O 334.3; 318.3; 319.3 -SO3; -SO3-H2O; GTX4 C10H17N7O9S 412 332; 314;253 318.3; 319.3; 338.3 -SO3-H2O-NH3-CO2 GTX2 C10H17N7O8S 396 316; 298 -SO3; -SO3-H2O; 322.1; 321.1; 305.1 -SO3; -SO3-H2O; GTX3 C10H17N7O8S 396 316; 298; 220 302.3; 303.3 -SO3-2H2O-NH3-NHCO
Toxins 2019, 11, 211 6 of 13 Table 2. Mass spectral data for gonyautoxins (GTX1-4). Molecular Fragment Ion after Toxin [M+H]+ Fragment Ion Loss of Formula Labeling GTX1 C10 H17 N7 O9 S 412 332; 314 -SO3 ; -SO3 -H2 O 334.3; 318.3; 319.3 -SO3 ; -SO3 -H2 O; GTX4 C10 H17 N7 O9 S 412 332; 314;253 318.3; 319.3; 338.3 -SO3 -H2 O-NH3 -CO2 GTX2 C10 H17 N7 O8 S 396 316; 298 -SO3 ; -SO3 -H2 O; 322.1; 321.1; 305.1 -SO3 ; -SO3 -H2 O; GTX3 C H N O S 396 316; 298; 220 302.3; 303.3 Toxins 2019, 11, 211 10 17 7 8 -SO3 -2H2 O-NH3 -NHCO 6 of 12 Toxins 2019, 11, 211 6 of 12 OCONH2 OCONH2 OCONH2 OCONH2 HO H HO H 6 N N 7H 6 HO N1 N HO N1 7H 6 6 N N1 7 N1 7 NH2+ NH2+ 2 9 NH2+ 2 9 NH2+ 3 3 2 N9 2 N9 +H2N N3 OH +H2N N3 OH HN HN +H2N N 12 H OH +H2N N 12 OH 10 OH H 12 10 12 OH 10 OH 10 OH 11 11 H 11 OSO3- -O3SO 11 H H OSO3- -O3SO H GTX1 GTX4 GTX1 GTX4 OCONH2 OCONH2 OCONH2 OCONH2 H H H H 6 N 6 N H N1 7H H N1 7H 6 N 6 N N1 7 N1 7 NH2+ NH2+ 2 9 NH2+ 2 9 NH2+ 3 3 2 N9 2 N9 +H2N N3 OH +H2 N N3 OH HN HN +H2N N 12 H OH +H2 N N 12 OH H 10 12 OH 10 12 OH 10 OH 10 OH 11 11 H 11 OSO3- -O3 SO 11 H H OSO3- -O3 SO H GTX2 GTX3 GTX2 GTX3 Figure 5. The molecular structural formula of GTX1-4. Figure 5. The molecular structural formula of GTX1-4. 334.2926 GTX1 GTX4 100 GTX1 100 GTX4 334.2926 318.3007 100 100 318.3007 80 80 80 80 290.2709 290.2709 60 60 60 60 %% %% 40 40 40 40 20 318.3007 20 20 291.2723 318.3007 362.3250 20 319.3054 291.2723 319.3054 362.3250 338.3418 319.3054 319.3054 338.3418 0 0 0 0 280 290 300 310 320 330 340 350 360 370 280 290 300 310 320 300 305 310 315 320 325 330 335 340 m/z 330 340 350 360 370 300 305 310 315 320 m/z 325 330 335 340 m/z m/z (a) (b) (a) (b) GTX2 GTX3 100 GTX2 100 302.3054 322.1197 GTX3 100 100 302.3054 322.1197 80 321.1252 321.1252 80 80 80 60 60 60 %% 60 %% 40 305.1583 40 305.1583 40 40 20 307.1704 317.0497 328.2077 20 20 307.1704 317.0497 328.2077 20 303.3095 346.3338 0 303.3095 346.3338 0 0 0 290 300 310 320 330 340 350 305 310 315 320 325 330 290 300 310 m/z 320 330 340 350 305 310 315 m/z 320 325 330 m/z m/z (c) (d) (c) (d) Figure6.6. Mass of 15 Mass spectrum of 15N-GTX4 15N-GTX1 (a), 15 N-GTX4(b), 15 N-GTX2 (b),1515 N-GTX2(c) and151515 (c)and N-GTX3 N-GTX3(d). Figure 6. Massspectrum Figure spectrum of 15N-GTX1 (a), 15N-GTX4 (b), N-GTX2 (c) and N-GTX3 (d). (d). 3. Discussion 3. Discussion Numerous studies have focused on the bioaccumulation and biotransformation of PSTs using Numerous studies have focused on the bioaccumulation and biotransformation of PSTs using Alexandrium strains, such as A. minutum in marine organisms [33]. Stable isotopes have been often Alexandrium strains, such as A. minutum in marine organisms [33]. Stable isotopes have been often used to study lipid synthesis, proteomics and ecosystem [13,15,34] and rarely applied to toxin-
Toxins 2019, 11, 211 7 of 13 3. Discussion Numerous studies have focused on the bioaccumulation and biotransformation of PSTs using Alexandrium strains, such as A. minutum in marine organisms [33]. Stable isotopes have been often used to study lipid synthesis, proteomics and ecosystem [13,15,34] and rarely applied to toxin-producing algae [35]. This study intends to use the biosynthetic process to replace the nitrogen atom of A. minutum with a stable isotope 15 N to form a tracer-enabled A. minutum for PST synthesis and metabolism. 3.1. Effect 15 N/P of on A. minutum Culture Growth and toxin production of toxic dinoflagellates vary with nutrients supply. High nutrient cultures can inhibit the photosynthetic capacity of photosystem II, which related to algae growth [27]. The N:P ratio can be used as an index for the nutritional status and physiological behavior of phytoplanktons [36]. Kinds of nitrogen sources and N:P supply ratio can affect the physiological responses of a tropical Pacific strain of A. minutum, the cellular toxin quota (Qt) was higher in P-depleted, nitrate-grown cultures [37]. To assure the feasibility of isotope 15 N, some experiments were carried out, including the comparison of different 15 N:P ratio. In batch culture, five 15 N:P ratios were compared, and the growth of A. minutum under 1.0 and 1.5 times of the f/2 medium standard 15 N/P concentration was better than others (Figure 1a). Similar previous findings reported that cell densities and growth rates of A. minutum were severely suppressed under high N/P ratios (>100) in both N-NO3 and N-NH4 treatments [38]. Some studies showed that the incorporation of 15 N-labeled salt did not affect the growth of green alga Chlamydomonas reinhardtii [24,39], and our study achieved the same result with A. minutum. The toxin profile of this A. minutum strain is relatively stable and predominantly constitutes GTX1-4 (Figure 3), the same as four strains of A. minutum collected from southern Taiwan [40], even under different N:P supply ratios. The highest algae toxicity per cell of all five groups was observed at day 25, and cellular toxin quota of the exponential growth phase was higher than that of the stable phase, even though the total number of stable cells is highest during the entire growth process (Figure 1b). The explanation could be that there was a negative correlation between algae toxicity per cell and cell density. In other words, the cell size was smaller when cell density was higher; thus, toxicity per cell of stable phase was lower. As previously reported, changes of nutrient availability with time in batch culture caused growth stage variability in toxin content, which peaked during mid-exponential growth [31]. Total toxicity, toxicity per cell, and the number of and relative proportion of toxin analogs changed in relation to the 15 N:P ratio. The f/2 medium standard 15 N/P concentration at 1.0 time was a better choice to label PSTs with 15 N regardless of cultivation method, i.e., batch culture or generation to generation culture, for A. minutum growth, PST production and profile, and experimental cost. 3.2. The Replacement of Stable Isotope 15 N The successful production PSTs of labeled substances from A. minutum was detected by MAT-271 Gas isotope mass spectrometer to determine 15 N abundance, Analysis by HPLC-MS was performed to identify 15 N atom number. In batch culture, δ15 N of two groups’ PSTs had a significant difference (p < 0.01) in different growth stages (lag, log, and stable phases) (Table 2). Combining 15 N labeling abundance with mass spectrum results, it can be presumed that artificial 15 N addition can bring 2~6 15 N atoms into the 15 N-PSTs molecule, and the 4~6 15 N atoms’ replacement becomes most possible. Recently, 15 N stable-isotope-labeling was applied to the toxic dinoflagellate Alexandrium catenella and the relationship between the order of 15 N incorporation % values of the labeled populations and the proposed biosynthetic route was established [35]. Relative abundance % of m+6 and m+7 isotopomers of PSTs were the highest in A. catenella after a two month passage in 15 N-NaNO3 medium in [35], which is similar to our conclusion. Nitrogen (N) isotopic compositions of PSTs in A. minutum cells reflect the isotopic fractionations associated with diverse biochemical reactions. Based on PSTs being a secondary metabolite, a small part of the supplied nitrogen was assimilated into PSTs, and most of
Toxins 2019, 11, 211 8 of 13 the nitrogen may participate in the synthesis of other nitrogen compounds. Some findings on high abundance in biomass (not extracted) have been reported; a process for the cost-effective production of 13 C/15 N-labelled biomass of microalgae on a commercial scale is presented, and 97.8% of the supplied nitrogen is assimilated into the biomass [23]. However, in the present paper, 15 N-labeling abundance of the 15 N-PSTs extract was 82.36%, lower than the abundance of whole cell in existing research [23]. The occurrence of this situation can be explained by the following reasons. Firstly, the time of each generation culture is not sufficiently long enough. Secondly, the total 15 N abundance in the crude extract of the toxin cannot fully represent 15 N in the pure toxin, because extract impurities (e.g., protein and pigment) can cause interference. Thirdly, trace amounts of nitrogen in natural seawater and the culture solution reagent may affect 15 N-labeled PSTs. 4. Conclusions This paper provides the initial evidence that 15 N isotope is feasible to label PSTs in A. minutum and worthy of being a powerful tool to conduct PST-related researches. In our study, 15 N abundance, PSTs content and profile were detected and the 15 N atom number introduced into GTX1-4 should be 4–6. However, it is a pity that the precursor and the biosynthetic intermediates of PSTs in A. minutum were not analyzed. Further study is needed to apply isotope-labeling on both toxin-producing algae and vector mollusk species so that we can better elucidate the mechanism of PSTs biosynthesis and metabolism. 5. Materials and Methods 5.1. Chemicals and Analytical Standards 15 N-NaNO 3 (δ15 N = 99.14%) was obtained from SRICI (Shanghai Research Institute of Chemical Industry CO., LTD., Shanghai, China). Bio-gel P-2 (400 mesh), Bio-Rex 70 (400 mesh) were obtained from BIO-RAD (Hercules, CA, USA). Certified reference materials for PSTs, including gonyautoxin 1/4 (GTX 1/4), gonyautoxin 2/3 (GTX 2/3), were purchased from the National Research Council, Institute for Marine Bioscience (Halifax, Canada). Analytical grade solvents were used for extraction purposes while LC grade solvents were used for HPLC-FLD applications. 5.2. Algal Culture The PSTs-producing dinoflagellate A. minutum (strain AGY-H46, purchased from Leadingtec, Shanghai, China) was cultivated in thermo regulated rooms (25 ± 1 ◦ C) with filtered (0.45 µm, Jinjing Ltd., China) and sterilized (121 ◦ C, 20 min) seawater before enrichment with f/2 medium amendments (Table 3). The light intensity was set at 3000–4000 lux with a dark:light cycle of 14:10 h. The seawater was obtained from Donghai Island waters (Zhanjiang, China). Algal cell densities were determined by optical microscope and cells were collected at particular time for 15 N abundance analysis and PSTs detection. Table 3. f/2 medium amendments. Reagent Working Solution (mg/L) Stock Solution (g/L) A: NaNO3 75 75 B: NaH2 PO4 ·H2 O 5 5 C: Na2 SiO3 ·9H2 O 20 20 D: Na2 EDTA 4.36 4.36 E: FeCl3 ·6H2 O 3.16 3.16 F: CuSO4 ·5H2 O 0.01 0.01 ZnSO4 ·7H2 O 0.023 0.023 CoCL2 ·6H2 O 0.012 0.012 MnCL2 ·4H2 O 0.18 0.18 Na2 MoO4 ·2H2 O 0.07 0.07
Toxins 2019, 11, 211 9 of 13 Table 3. Cont. Reagent Working Solution (mg/L) Stock Solution (g/L) G: Vitamin B1 0.1 0.01 Vitamin B12 0.5 × 10−3 0.5 × 10−4 Vitamin H 0.5 × 10−3 0.5 × 10−4 5.3. 15 N-PSTs Extraction An aliquot (60 mL) of the algal fluid was centrifuged at 6000 r/min under 4 ◦ C for 10 min, and the supernatant was carefully discarded. The sedimentary cells were resuspended with 0.05 M acetic acid and then broken using ultrasonic processor in an ice bath for 10 min (power 80%, working 3 s, gap 3 s) until there was no whole cell. The combined liquid was centrifuged at 12000 r/min under 4 ◦ C for 10 min, then the supernatant was filtered (0.22 µm, Jinjing Ltd., China) and stored under −20 ◦ C for purification. 5.4. 15 N-PSTs Separation and Purification Separation and purification were carried on by reference to published papers [41,42]. The PSTs extract was adsorbed to a column of Bio-Gel P-2 equilibrated with water. Furthermore, the column was first washed with a sufficient volume of water and eluted with 0.1 mM acetic acid at a flow rate of 0.5 mL/min. After separation, toxin mixture was purified by ion exchange chromatography using a column of Bio-Rex 70 equilibrated with water and gradient eluted with acetic acid (acetic acid concentration was as follows: 0, 0.05, 0.055 and 0.060 mM). The fraction was collected every 12 min and then analyzed by FFA to assure purification efficiency. 5.5. 15 N Abundance Analysis of 15 N-PSTs Extracts 15 N abundance was analyzed by MAT-271 Gas isotope mass spectrometer (Finnigan, Santa Clara Valley, CA, USA). Operating conditions were set to high voltage (10 kV), emission current (0.040 mA), electronic energy (100 eV) and well voltage (134 eV). 15 N-PSTs extraction after freeze-drying was converted to gas by micro-high-heat combustion method, then entered into gas isotope mass spectrometer sample introduction system via sample adapter. Mass spectrometer vacuum was less than 2 × 10−5 Pa. After making the necessary calibration settings for the instrument, the instrument background measurement was performed. The sample gas was introduced into the sample storage system at a pressure of 5–10 Pa. The measurement process was automatically performed by computer instructions, and the signal strength (in mV) of mass number 28, 29, 30 was output as I28 , I29 and I30 . 5.6. PSTs Toxicity Test by the Mouse Bioassay The mouse bioassay (MBA) was a referenced method from [43]. 10 healthy male mice were injected intraperitoneally with 1 mL aliquot of 15 N-PSTs extracts and observed to quantify the toxin according to the time of death. The toxicity was expressed in mouse units (MU), 1 MU representing the average toxin amount to kill a mouse weighing 20 g within 15 min. 5.7. 15 N-PSTs Detection by the Fast Fluorimetric Assay (FFA) The fast fluorimetric assay (FFA) was performed by fluorospectrophotometer (HITACHI, Japan) at a 333 nm excitation wavelength, 10 nm excitation slit, emission wavelength 390 nm and 20 nm emission slit. The method was from [44] and got some modification in this paper. A portion (0.5 mL) of each extract or blank solution (0.05 M acetic acid), respectively, was mixed with 2 mL of oxidation solution (50 mM dipotassium phosphate with 10 mM periodic acid) and incubated for 15 min at 50 ◦ C. After incubation, the reaction mixture was neutralized with 2.5 mL of 1 M acetic acid and transferred to a cuvette for detection by the fast fluorimetric assay (FFA). Relative fluorescence units
Toxins 2019, 11, 211 10 of 13 (RFU) was recorded and this experiment was conducted to get an overview of the fluorescence of oxidized samples, but not for accurate quantification. 5.8. 15 N-PSTs Determination by HPLC-FLD HPLC-FLD analysis was carried out using Agilent 1100 (Agilent, Santa Clara, CA, USA) coupled to PCX5200 (Pickering Laboratories Company) post-column reactor and Agilent G1321A detector (λex = 330 nm, λem = 390 nm). Chromatographic separation of compounds was achieved on a reversed-phase C8 column (150 mm × 4.6 mm i.d.; 5 µm, GL Sciences, Tokyo, Japan). The system was managed by an Agilent Chem. Station 2.1 workstation. The PST extract was subjected to HPLC-FLD after ultrafiltration (10,000 Da ultrafiltration centrifuge tube, 12,000 r/min for 10 min at 4 ◦ C). Analysis method was performed according to [45]. The oxidant solution was 50 mM potassium hydrogen phosphate containing 7 mM periodic acid. The acidifying agent was 0.5 M acetic acid. The flow rates were 0.8 mL/min for the elution solution and 0.4 mL/min for the oxidant and acidifying agent. 5.9. 15 N-PSTs Quantification by HPLC-MS HPLC-MS analysis was carried out on Agilent 1100 (Agilent, USA) coupled to Waters FIN_C2-XS QTof mass spectrometer (Waters, Milford, MA, USA) with an electrospray ionization interface. PSTs were separated on a TSKgel Amide-80 HILIC column (250 mm × 2 mm i.d.; 5 µm, Tosoh Bioscience, LLC, Montgomeryville, PA, USA). The PSTs extract after purification was subjected to HPLC-MS after ultrafiltration (10000 Da ultrafiltration centrifuge tube, 12000 r/min for 10 min at 4 ◦ C). A binary mobile phase included “solvent A” and “solvent B”, in which “solvent A” was water containing 2.0 mM ammonium formate containing 0.1% (v/v) formic acid and “solvent B” was acetonitrile containing 0.1% (v/v) formic acid. Parameters of mass spectrometer were multiple reaction monitoring (MRM) mode, positive polarity of ESI, capillary voltage (3.2 kV), ion source temperature (150 ◦ C), desolvation temperature (400 ◦ C), cone gas flow (50 L/h) and desolvation gas flow (700 L/h). Author Contributions: Methodology, X.Y.; investigation, M.L., R.Z. and X.H.; writing-original draft preparation, M.L.; writing-review and editing, W.X.; supervision, L.S., and C.L.; project administration, W.X. and X.Y. Funding: This research was funded by National Natural Science Foundation of China, grant number 31271938 and 31772089, and Shandong Province Key Research and Development Program, grant number 2017GHY15127. This work was also financially supported by Major Scientific Research Projects in Universities Cultivation Plan (Q15137) GDOU2015050202. Acknowledgments: This research was funded by National Natural Science Foundation of China, grant number 31271938 and 31772089, and Shandong Province Key Research and Development Program, grant number 2017GHY15127. This work was also financially supported by Major Scientific Research Projects in Universities Cultivation Plan (Q15137) GDOU2015050202. We gratefully acknowledge the help of the other members in our lab, who have offered us valuable suggestions in the academic studies. Besides, we also show our gratitude to Shanghai Research Institute of Chemical Industry CO., LTD., which provided technical support for us. Conflicts of Interest: The authors declare no conflict of interest. References 1. Hoagland, P.A.; Anderson, D.M.; Kaoru, Y.; White, A.W. The Economic Effects of Harmful Algal Blooms in the United States: Estimates, Assessment Issues, and Information Needs. Estuaries 2002, 25, 819–837. [CrossRef] 2. Gustaafm, H. Ocean climate change, phytoplankton community responses, and harmful algal blooms: A formidable predictive challenge. J. Phycol. 2010, 46, 220–235. [CrossRef] 3. Rolton, A.; Vignier, J.; Soudant, P.; Shumway, S.E.; Bricelj, V.M.; Volety, A.K. Effects of the red tide dinoflagellate, Karenia brevis, on early development of the eastern oyster Crassostrea virginica and northern quahog Mercenaria mercenaria. Aquat. Toxicol. 2014, 155, 199–206. [CrossRef] [PubMed] 4. Brooks, B.W.; Lazorchak, J.M.; Howard, M.D.; Johnson, M.V.; Morton, S.L.; Perkins, D.A.; Reavie, E.D.; Scott, G.I.; Smith, S.A.; Steevens, J.A. Are harmful algal blooms becoming the greatest inland water quality threat to public health and aquatic ecosystems? Environ. Toxicol. Chem. 2016, 35, 6–13. [CrossRef] [PubMed]
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